Induced pluripotent stem (iPS) cells represent a powerful source for cell-based tissue regeneration because they are patient-specific cells and can differentiate into specialized cell types. Previously, we have demonstrated the derivation of neural crest like cells from iPS cells (iPS-NCLCs), and these cells have the potential to differentiate into dental mesenchymal cells, which subsequently differentiate into odontoblasts and dental pulp cells. In this study, we show that iPS-NCLCs can differentiate into mesenchymal stem cells (iPS-NCLC-MSCs), which contribute to craniofacial bone regeneration. iPS-NCLCs were cultured in serum-containing media and differentiated into functional MSCs, as confirmed by expression MSC markers and their ability to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro. iPSNCLC- MSCs were negative for markers of undifferentiated iPS cells and did not develop into teratomas when transplanted to immunodeficient mice. Further, iPS-NCLC-MSCs grew normally and differentiated into osteoblasts on hydroxyapatite scaffolds in vitro. To assess the potential of iPS-NCLC-MSCs to regenerate craniofacial bone in vivo, iPS-NCLC-MSCs were transplanted into critical-size calvarial defects in immunodeficient mice for 8 weeks. Histological analysis revealed that iPS-NCLC-MSCs differentiated into osteoblasts and contributed to bone regeneration without tumor formation. These results indicate that iPS-NCLC-MSCs could be a potential candidate for cell-based craniofacial bone tissue repair and regeneration.
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For clinical application of pluripotent stem cells in tissue regeneration, tumorigenesis must be the most taken into account. In the present study, we proved that iPS-NCLC-MSCs had no tumorigenicity in vivo. The safety of iPS-NCLC-MSCs further confirmed the reduction of some pluripotency genes as shown in our previous study of iPS-NCLCs12). Consistent with our results, recent studies related to MSCs derived from iPS cells have demonstrated their safety for clinical application32,34-36), providing evidence to promote the use of iPS-NCLC-MSCs for stem cell therapy. Interestingly, in iPS-NCLC-MSCs in the present study, Sox2 expression level was unchanged compared to that for undifferentiated iPS cells. Sox2 not only regulates the self-renewal of ESCs and plays an important role in the maintenance of adult Shintaro Sukegawa et al.: Clinicopathology of Japanese Ameloblastic Fibro-Odontoma stem cells, but also closely relates to many types of cancer in several 2. Arrington ED, Smith WJ, Chambers HG, Bucknell AL and Davino tissues such as lung, esophagus, and breast37). Sox2 also maintains the NA. Complications of iliac crest bone graft harvesting. Clin Orthop capability for expansion and differentiation of MSCs38). However, the Relat Res 329: 300-309, 1996 role of Sox2 in the tumorigenesis of iPS cells is still unclear. In addition Joshi A and Kostakis G. An investigation of post-operative to Sox2, the relationship between other genes and tumorigenesis has morbidity following iliac crest graft harvesting. Br Dent J 196: 167-been under debate. Zou et al. found that iPS-derived MSCs were 171, 2004 positive for Nanog despite the exclusion of other pluripotent markers34). Kainer MA, Linden JV, Whaley DN, Holmes HT, Jarvis WR, Lee et al. documented that polysialic acid-neural cell adhesion Jernigan DB and Archibald LK. Clostridium infections associated molecule (PSA-NCAM)-negative NC cells derived from human ESCs with musculoskeletal-tissue allografts. N Eng J Med 350: 2564-were tumorigenic39). These findings suggest the importance and further 2571, 2004 necessity of basic and preclinical studies to evaluate the safety of the Parekkadan B and Milwid JM. Mesenchymal stem cells as use of iPS cell-derived MSCs. therapeutics. Annu Rev Biomed Eng 12: 87-117, 2010 We further explored the possibility of clinical use of iPS-NCLC-Kawano S, Otsu K, Kuruma A, Shoji S, Yanagida E, Muto Y, MSCs for craniofacial bone regeneration. First, we evaluated the Yoshikawa F, Hirayama Y, Mikoshiba K and Furuichi T. ATP engraftment and differentiation capacity of iPS-NCLC-MSCs on HA, autocrine/paracrine signaling induces calcium oscillations and the major mineral component of bone, and demonstrated that they NFAT activation in human mesenchymal stem cells. Cell Calcium exhibited the typical morphology of MSCs and could differentiate 39: 313-324, 2006 into osteoblasts by osteogenic induction. These results coincided Ozawa K, Sato K, Oh I, Ozaki K, Uchibori R, Obara Y, Kikuchi with a previous study showing that MC3T3-E1 cells grew well on Y, Ito T, Okada T and Urabe M. Cell and gene therapy using the UDPHAp surface20) and suggested that iPS-NCLC-MSCs could mesenchymal stem cells (MSCs). J Autoimmun 30: 121-127, 2008 maintain the differentiation capacity into osteoblasts on HA when Stenderup K, Justesen J, Clausen C and Kassem M. Aging is transplanted. Second, we transplanted iPS-NCLC-MSCs with β-TCP associated with decreased maximal life span and accelerated in a calvarial defect and showed that the cells contributed to bone senescence of bone marrow stromal cells. Bone 33: 919-926, 2003 regeneration with no formation of tumor in vivo. In the implants, iPS-Kretlow JD, Jin Y-Q, Liu W, Zhang WJ, Hong T-H, Zhou G, Baggett NCLC-MSCs expressed osteoblast marker genes, indicating that the LS, Mikos AG and Cao Y. Donor age and cell passage affects cells could differentiate into osteoblasts and directly contribute to differentiation potential of murine bone marrow-derived stem cells. bone regeneration in vivo. Recent reports have also shown that adult BMC Cell Biol 9: 60, 2008 MSCs have the capacity to secrete factors such as soluble extracellular 10. Wagner W, Bork S, Horn P, Krunic D, Walenda T, Diehlmann A, matrix glycoprotein, cytokines, and growth factors to modulate the Benes V, Blake J, Huber F-X, Eckstein V, Boukamp P and Ho AD. microenvironment and recruit resident cells to repair the degenerated Aging and replicative senescence have related effects on human tissue40). Thus, transplanted iPS-NCLC-MSCs in this study may also stem and progenitor Cells. PLoS One 4: e5846, 2009 play a role in activating endogenous osteogenic progenitor cells by 11. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, secretion of such factors. Mosca JD, Moorman M A, Simonetti DW, Craig S and Marshak Here, we showed the efficient differentiation protocol from iPS cells DR. Multilineage potential of adult human mesenchymal stem into MSCs via NC induction, and that the iPS-NCLC-MSCs construct cells. Science 284: 143-147, 1999 is promising to promote bone regeneration during calvarial bone 12. Otsu K, Kishigami R, Oikawa-Sasaki A, Fukumoto S, Yamada A, repairs. Further study in other craniofacial implantation models such as Fujiwara N, Ishizeki K and Harada H. Differentiation of induced periodontal and jawbone defects is required to validate its efficiency. pluripotent stem cells into dental mesenchymal cells. Stem Cells Also, the functionality for vascularization and mineralization to form Dev 21: 1156-1164, 2012 better bone as well as the safety issues of tumorigenicity and immune 13. Otsu K, Kumakami-Sakano M, Fujiwara N, Kikuchi K, Keller L, rejection should be carefully evaluated in the long term. In addition to a Lesot H and Harada H. Stem cell sources for tooth regeneration: tissue regeneration standpoint, as bone formation by iPS-NCLS-MSCs current status and future prospects. Front Physiol 5: 36, 2014 in this study follows the developmental steps of craniofacial bone, this 14. Morikawa S, Mabuchi Y, Niibe K, Suzuki S, Nagoshi N, Sunabori could be used to aid in the understanding of normal craniofacial bone T, Shimmura S, Nagai Y, Nakagawa T and Okano H. Development development mechanisms and become a powerful tool for disease of mesenchymal stem cells partially originate from the neural crest. modeling and drug discovery. Biochem Biophys Res Commun 379: 1114-1119, 2009 15. Takashima Y, Era T, Nakao K, Kondo S, Kasuga M, Smith AG, Acknowledgements and Nishikawa S-I. Neuroepithelial cells supply an initial transient This work was financially supported by JSPS KAKENHI Grant wave of MSC differentiation. Cell 129: 1377-1388, 2007 Numbers 24659819 and 21592500, JSPS Bilateral Joint Research 16. Isern J, García-García A, Martín AM, Arranz L, Martín-Pérez Projects (2013-2015), Open Research Project Grant (2007–2011) from D, Torroja C, Sánchez-Cabo F and Méndez-Ferrer S. The neural MEXT, and a Grant from Keiryokai Research Foundation No. 118. crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function. Elife 3: e03696, 2014
This work was financially supported by JSPS KAKENHI Grant Numbers 24659819 and 21592500, JSPS Bilateral Joint Research Projects (2013-2015), Open Research Project Grant (2007-2011) from MEXT, and a Grant from Keiryokai Research Foundation No. 118.
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All Science Journal Classification (ASJC) codes
- Medicine (miscellaneous)
- Orthopedics and Sports Medicine
- Cell Biology